Fig 2: TE signatures during the primed-to-naive transition.a EPI, TE, PrE and trophoblast stem cell (TSC) signature scores of the primed-to-naive transitioning intermediates. b PCA of the bulk RNA-seq datasets (circles) from the primed-to-naive transitioning intermediates with published RNA-seq (diamonds) datasets32. n ≥ 2. c Expression of GATA3 in FDL. d Experimental design for the induction of TSCs from the primed-to-naive intermediates. e TP63 and KRT7 immunostaining of TSCs derived from day 8 RFP− and day 8 RFP+ cells during the primed-to-naive transition. Scale bars, 20 μm. Representative images from n = 3. f PCA of the bulk RNA-seq datasets (circles) from the transitioning intermediates-derived TSCs with published RNA-seq (diamonds) datasets32. n ≥ 2. g Heatmap (left) and TSC score (right) showing the expression levels of representative TSC-related genes during the TSC derivation process from RFP− and RFP+ transitioning intermediates on day 8. Source data are provided as a Source Data file. h HLA-G, SDC1 and CGB immunostaining of extravillous trophoblast (EVT) (upper) and syncytiotrophoblast (ST) (lower) cells, respectively. EVT and ST cells were differentiated from day 8-RFP− and day 8-RFP+ cell-derived TSCs. Scale bar, 20 μm. Representative images from n = 3. i Representation of day 8-RFP− and day 8 RFP+ cell-derived TSC engraftment assay by injection into NOD-SCID mice. j Immunostaining of TP63, HLA-G and SDC1 in the lesions collected from day 8-RFP− and day 8 RFP+ cell-derived TSC engrafts in NOD-SCID mice. No lesions were evident in the vehicle controls. Scale bar, 20 μm. Representative images from n = 3. k Representative positive results for the hCG pregnancy test performed on urine samples, serum samples, and ST cell culture supernatant collected from day 8-RFP− cell -derived TSCs. Source data are provided as a Source Data file.

Fig 3: The C-terminus of p63 protein is required for the ability to inhibit R-mediated lytic reactivation.NOKs-Akata cells were transfected with or without an R expression vector in the presence or absence of various different co-transfected p63 isoform vectors, including the human ΔNp63ɑ protein, the mouse ΔNp63ɑ protein, the mouse ΔNp63β isoform (homologous to ΔNp63ɑ but lacking 121 amino acids at the C-terminal domain), or the mouse TAp63α isoform (which is identical to ΔNp63ɑ except for the addition of an N terminal transactivating domain). Two days after transfection, immunoblots were performed to assess expression levels of the Z, R, BMRF1, p63 protein isoforms, and tubulin as indicated. The quantification of Z protein expression was determined through ImageJ, normalized to tubulin, with the R alone condition set as 1. The black lines show where irrelevant lanes were removed; the original western blots used to construct this figure are shown in supplemental Fig 7. This experiment is representative of two independent biological replicates.

Fig 4: Quantitative analysis of cell marker expression by epithelial cells from each cervical region.Freshly isolated cells from each cervical region were stained with antibodies against K14, K18, K17, p63 or MMP7. Bars represent the mean ± standard error of three experiments using samples from different donors. The asterisks indicate statistical differences [** P < 0.01;*** P < 0.001].

Fig 5: ARID1A loss relieves Atf3 repression associated with squamous differentiation.a, Representative H&E histology of (from left to right) control mice, TP53/PIK3CA mutants, ARID1A/PIK3CA mutants, and TP53/ARID1A/PIK3CA mutants. Arrowheads depict endometrial epithelia. b-d, Immunohistochemical staining of ATF3 (b), TP63 (GeneTex) (c), and COL17A1 (d) of mouse sections as in a.